Abstract

De novo DNA methylation in plants relies on transcription elongation of RNA polymerase V (Pol V) along with KTF1 and SPT4, which produces long non-coding RNAs for recruitment and assembly of DNA methylation machinery. Here, we report a cryo-EM structure of the KTF1-bound Pol V transcription elongation complex. The structure reveals conformation of the structure motifs in the active site of Pol V that accounts for its inferior RNA-extension ability. The structure further reveals structural features of Pol V that prevent it from interacting with the general transcription factors of Pol II and Pol IV. The KOW5 domain of KTF1 binds near the RNA exit channel of Pol V, where it provides a scaffold for the proposed recruitment of Argonaute proteins to initiate the assembly of DNA methylation machinery. The structure provides insight into the Pol V transcription elongation and the role of KTF1 during Pol V transcription-coupled DNA methylation.